Over the years, the focus of this grant has been on the regulation of segmentation genes and homeotic genes, including even-skipped (eve) and Abdominal-B (Abd-B). However, the early Drosophila embryo offers a number of outstanding opportunities for systems level studies in developmental biology, such as quantitative modeling and whole-genome assays. During the past few years we have attempted to use ChIP-chip and ChIP-seq assays along with quantitative methods to achieve a more global view of gene regulation in the Drosophila embryo. These studies led to a few surprising findings, including the occurrence of stalled or paused Pol II in the promoter regions of many or most developmental control genes, and the identification of shadow enhancers--remote copies of an enhancer that were missed by conventional gene fusion assays. During the upcoming funding period we will attempt to determine the significance of these new findings. We will use quantitative in situ hybridization methods to examine the induction of stalled and non-stalled genes in the early embryo to determine whether they display distinctive activities. We will also compare the responses of genes that contain or lack shadow enhancers to genetic perturbation. In particular, are genes with shadow enhancers better buffered to changes in gene dose than comparable genes lacking shadows? Finally, we are keen to apply ChIP-seq and chromosome conformation capture (3C) assays to understand the basis for short-range transcriptional repression. For example, do such repressors block enhancer-promoter interactions by precluding the recruitment of chromosomal coactivators such as p300/ CBP? The research plan is composed of three specific aims: 1) quantitative confocal image analysis of stalled and nonstalled developmental patterning genes; 2) the use of 3C (chromosome conformation capture) and ChIP-seq assays to determine whether short- range transcriptional repressors inhibit enhancer-promoter interactions; and 3) determine the role of noncoding RNAs, such as bxd, in the regulation of Ubx and other Hox genes.